Enteropathogenic Escherichia coli (EPEC) are a significant cause of infantile diarrhea. The long-term objectives of this project are to study the pathogenesis of disease due to this organism and to develop DNA probes for diagnosis and vaccine candidates for prevention. Recent evidence indicates that the pathogenesis of EPEC is a two-stage process. Stage 1 involves plasmid-mediated adherence factors which are responsible for the intimate adherence of the bacteria to the microvilli resulting in the characteristic EPEC histopathology. The features of the EPEC lesion include effacement and dissolution of the brush border along with dense condensation of actin microfilaments in the enterocyte cytoplasm directly beneath the adherent bacterium. Similar to the multiple possible colonization factors in human and animal enterotoxigenic E. coli, there appear to be multiple factors responsible for stage 1 adherence in EPEC. At least two such factors (class I and class II adhesins) are found in strains pathogenic for humans and a third factor may be present in strains pathogenic for rabbits (RDEC-1). The chromosomal factor responsible for stage 2 brush border effacement and actin condensation (Actin Condensation Factor, ACF) appears to be common for all EPEC. The primary goal of the proposed research is to investigate the pathogenesis of EPEC using molecular genetic techniques. The genes encoding the ACF will be cloned, sequenced, and mutated and the gene products and regulation of genes encoding the plasmid-borne class I adhesin will be studied. A DNA probe to detect the ACF will be developed and evaluated using isolates from an ongoing epidemiological study of infant diarrhea in Santiago, Chile. The genes encoding the class II adhesin will be mutated and a DNA probe developed and evaluated for this factor. The interaction of plasmid and chromosomal virulence factors will be studied using tissue culture and rabbit models. Finally, attenuated vaccine candidates will be developed for the class I EPEC by deleting the genes for the ACF while retraining the genes for the initial class I adhesions.